Candida albicans is the leading fungal pathogen in normal hosts and in patients with damaged immune systems. In normal hosts, disease caused by C. albicans ranges from mild, easily treated, superficial disease (e.g., thrush in newborn infants; paronychia in workers whose hands are immersed in water) to more severe, chronic or recurrent infections (e.g., candidal vaginitis). It is estimated that 5% of women of child-bearing age will suffer from recurrent candidal vaginitis (Hurley, xe2x80x9cTrends in candidal vaginitis.xe2x80x9d Proc. R. Soc. Med. 70 (Suppl. 4), 1-8 (1970), and that virtually every woman will experience at least one episode during her reproductive years. Vaginitis is particularly frequent in otherwise normal females with diabetes or a history of prolonged antibiotic or oral contraceptive use. While short-term topical therapy is effective in treating individual episodes of vaginitis, such agents do not prevent recurrences. Thus, even in the normal host, infection with C. albicans can occur at epithelial surfaces, and recurrences are not prevented by presently available therapies.
In immunocompromised hosts such as cancer patients, transplant patients, post-operative surgical patients, premature newborns, or HIV-infected people, C. albicans ranks as the leading fungal pathogen. In this population, disease ranges from aggressive local infections such as periodontitis, oral ulceration, or esophagitis in HIV-infected patients, to complex and potentially lethal infections of the bloodstream with subsequent dissemination to brain, eye, heart, liver, spleen, kidneys, or bone. Such grave prognoses require more toxic therapy, with attendant consequences from both the underlying infection and the treatment. Here again, the infection typically begins at an epithelial site, evades local defenses, and invades the bloodstream in the face of immunosuppression. Strategies to interrupt candidal adhesion therefore have broad applicability to the prevention of mild but recurrent disease in the normal host and to the reduction of substantial morbidity and mortality in the immunocompromised.
It is well recognized that C. albicans adheres to epithelial and endothelial cells in the human host, oftentimes by recognizing proteins of the extracellular matrix called ligands. These ligands include proteins such as fibronectin, vitronectin, fibrinogen, the C3 degradation fragment iC3b, or the shorter C3 degradation fragment C3d. Because recognition of all of these proteins except C3d is dependent upon the amino acid sequence ARGININE-GLYCINE-ASPARTIC ACID or R-G-D, these candidal adhesions are thought to operate like the vertebrate integrins and are called xe2x80x9cintegrin-like proteinsxe2x80x9d or xe2x80x9cintegrin analogs.xe2x80x9d
Vertebrate integrins are composed of two subunits: an xcex1-subunit and a xcex2-subunit. There are approximately 14 xcex1 and 8 xcex2 subunits described to date in vertebrate cells. Using monoclonal or polyclonal antibodies to vertebrate integrins, several investigators have obtained evidence for integrin-like proteins in C. albicans: an xcex1M analog, an xcex1 5/xcex21 complex, or a xcex21 analog. Neither the xcex1 5/xcex21 complex nor the xcex21 analog has been isolated from C. albicans or from any other candidal species, and the responsible genes encoding these xe2x80x9cintegrin-like proteinsxe2x80x9d have not been identified.
The present invention provides an isolated and purified DNA molecule encoding a Candida albicans protein with integrin-like motifs that hybridizes to DNA complementary to DNA having SEQ ID NO:1 under the stringency conditions of hybridization in buffer containing 5xc3x97SSC, 5xc3x97Denhardt""s, 0.5% SDS, 1 mg salmon sperm/25 mls of hybridization solution incubated at 65xc2x0 C. overnight, followed by high stringency washing with 0.2xc3x97SSC/0.1% SDS at 65xc2x0 C. Preferably, the present invention provides an isolated and purified DNA molecule encoding the Candida albicans protein with integrin-like motifs which has the amino acid sequence having SEQ ID NO:2. Preferably, the DNA is genomic DNA which has the nucleotide sequence shown in Table 1 (SEQ ID NO:1).
The present invention also provides a vector and a cell line transformed by an extrachromosomal plasmid containing non-native DNA encoding Candida albicans protein with integrin-like motifs (i.e., C. albicans integrin-like protein), as described herein. The cell line preferably comprises S. cerevisiae. This cell line can be used in a method of delivering a gene product to a subject.
The present invention also provides a Candida albicans protein with integrin-like motifs comprising an I domain, two EF-hand divalent cation binding sites, a sequence sufficient to encode a transmembrane domain, an internal RGD tripeptide, and a carboxy-terminal sequence with a single tyrosine residue. As used herein, an xe2x80x9cinternalxe2x80x9d RGD tripeptide means that the RGD sequence is in the Candida protein, not in the vertebrate proteins recognized by integrins. Preferably, the isolated and purified C. albicans integrin-like protein has an amino acid sequence which is SEQ ID NO:2. Also provided are isolated and purified peptides, such as those having an amino acid sequence selected from the group consisting of: YLS PTN NNN SKN VSD MDL HLQ NL (SEQ ID NO:4); DWK LED SND GDR EDN DDI SRF EK (SEQ ID NO:5); SKS ANT VRG DDD GLA SA (SEQ ID NO:6); DHL DSF DRS YNH TEQ SI (SEQ ID NO:7); and WIQ NLQ EII YRN RFR RQ (SEQ ID NO:8). The invention also provides a vaccine comprising the protein and peptides, either singly or together, described herein as well as an isolated and purified antibodies to the C. albicans integrin-like protein and peptides described herein.
The invention also provides a method of inhibiting adhesion of Candida albicans to cells (preferably epithelial cells, and more preferably human epithelial cells). The method includes contacting the Candida albicans with antibodies to the Candida albicans protein with integrin-like motifs (xcex1Int1p) or to fragments thereof as described herein.